The present invention is related to, but not limited to, enzymes for cleaving α2-antiplasmin, and inhibitors thereof, and to screening methods for identifying such inhibitors and to methods for treating conditions involving fibrin.
Plasmin plays a critical role in fibrin proteolysis and tissue remodeling (1). Among plasmin inhibitors, α2-antiplasmin (α2AP) is the most important (2). The physiological relevance of plasmin inhibition by α2AP to blood clotting and fibrinolytic homeostasis is supported by the following observations: (i) plasma α2AP inactivates circulating free plasmin in about 0.1 second (1), thereby eliminating the potential for a systemic lytic state that could lead to bleeding; (ii) α2AP is crosslinked to forming fibrin by activated blood clotting factor XIII (FXIIIa) and inhibits plasmin-mediated lysis in direct proportion to the amount incorporated (3,4); (iii) patients with homozygous α2AP deficiency manifest serious hemorrhagic tendencies, while heterozygotes tend to hemorrhage only after major trauma or surgery (5); (iv) two adult patients with relatively large patent ducti arteriosi (PDA) were successfully treated by continuous intravenous α2AP to stabilize induced thrombi within the PDA until completely occlusive and ready for fibrous organization (6); and (v), in mice homozygous for targeted α2AP gene disruption (α2AP-/-), experimental induction of thrombi took longer, bleeding times were prolonged, and vascular patency was more readily established when compared with wild-type mice (7). The sum of these observations underscores the importance of α2AP on the progression and survival of physiologic and pathologic fibrin formation.
α2-Antiplasmin (α2AP) is therefore a blood plasma protein that rapidly and specifically inhibits the enzyme, plasmin, which digests blood clots, whether presenting early as intravascular platelet-fibrin deposits or as partially or completely occlusive thrombi. Similarly, plasmin and α2AP activities are important to the development and survival of fibrin as occurs in inflammation, wound healing and virtually all forms of cancer and its metastases. Human α2AP is synthesized primarily in the liver and secreted into plasma as a 68-kDa single polypeptide chain having Met as the N-terminus and containing 464 amino acids and 13% carbohydrate (1,8). During circulation in plasma, the secreted precursive Met-α2AP form undergoes proteolytic cleavage between Pro12-Asn13 to yield a truncated version with Asn as the new N-terminus, i.e., Asn-α2AP (8). The latter becomes crosslinked to fibrin about 3-fold faster than recombinant Met-α2AP (9). Despite the likelihood that this observation is physiologically important, an enzyme responsible for this cleavage previously has been unknown.
It was therefore a desirable objective to isolate and identify the plasma enzyme responsible for the cleavage of α2-antiplasmin into the active form.